Water-unextractable Arabinoxylan Research Articles

A screening method for endo-β-1,4-xylanase substrate selectivity

Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.

Refrigerated dough syruping in relation to the arabinoxylan population.

Refrigerated doughs develop syruping upon prolonged storage. To assess the role of arabinoxylans (AX), in this phenomenon, the evolution of the AX population and syruping in refrigerated doughs during storage were studied. When doughs were kept at 6 degrees C for up to 34 days of storage, dough syruping increased from 0% (fresh dough) to 22% of dough weight, reaching a plateau after 16 days of storage. High-performance size exclusion chromatography and gas-liquid chromatography showed hydrolysis of water-unextractable AX in the refrigerated dough, resulting in increased levels of solubilized AX in the first 2 days of storage. Longer storage resulted in further degradation of solubilized and water-extractable AX. Increased syruping was accompanied by a decrease in farinograph dough consistency. The results support the hypothesis that loss of water-holding capacity due to degradation of AX by endogenous xylanases is responsible for dough syruping.

Effects of genotype and harvest year on content and composition of dietary fibre in rye (Secale cereale L) grain

AbstractThe aim of this study was to measure the variation in dietary fibre (DF) content and composition among different rye varieties grown in the same location in three successive years and to estimate the contributions of genotype and harvest year to this variation. The study included grain from 19 different varieties of rye, of which seven varieties were analysed in all three years. The content of total DF in all samples ranged from 147 to 209 g kg−1 dry matter (dm), with 34–66 g kg−1 being water‐extractable (WE) DF and 108–159 g kg−1 being water‐unextractable (WUE) DF. The main DF component was arabinoxylan (AX) with a content of 80–121 g kg−1 dm, of which 26–41 g kg−1 dm was WE AX. The ratio of arabinose to xylose (ara/xyl) of total AX ranged from 0.59 to 0.69. The content of β‐glucan was 13–22 g kg−1 dm and that of fructan was 45–64 g kg−1 dm. Analysis of the data from seven varieties and three harvest years showed a highly significant influence of harvest year on most DF components. Significant genotype effects were found for the contents of total DF, total AX, β‐glucan and fructan and for the ara/xyl ratio of total and WUE AX. Estimation of variance components showed that yearly variations in the contents of total DF, total AX, WUE DF, WUE AX and β‐glucan were generally higher (27–55% of total variance) than genotype effects (14–19%). The ara/xyl ratio of total and WUE AX showed a high influence of genotype (46 and 43% respectively). Non‐adapted rye varieties with small kernel size had the highest contents of total DF and total AX. The content of β‐glucan was positively correlated with kernel weight (r = 0.68), whereas no overall correlation was found between kernel size and the content of DF. As a conclusion, the variation in DF content and composition in this study was significantly influenced by both harvest year and rye genotype.© 2002 Society of Chemical Industry

Structural Characterisation of Water-extractable and Water-unextractable Arabinoxylans in Wheat Bran

Abstract Water-extractable arabinoxylan (WE-AX) in wheat bran, of which only a minority originates from adherent endosperm, amounted to 6% of the total arabinoxylan (AX) content in such bran. WE-AX had an arabinose to xylose (A/X)-ratio of 0·45. Graded ethanol precipitation (20–80% range) yielded AX with an A/X-ratio increasing from 0·31 to 0·85. A population of molecular weight (MW) of 50 kDa precipitated between 0 and 40% ethanol and one with much lower apparent MW precipitated at higher ethanol concentrations. Wheat bran unextractable cell wall material (UCM) was obtained as the residue withstanding thermostable α-amylase and protease treatments and consisted mainly of AX (ca 45%) and cellulose (30–35%). Two consecutive extractions of UCM with alkaline hydrogen peroxide (AHP; 2·0%, pH 11·5, 4 h, 60 °C) resulted in a cellulose rich residue (CRR) containing 33% of the AX originally present in UCM. The average A/X-ratio of AX in CRR was lower than that in UCM. The extracted AX polymers (ca 45% of the AX originally present in UCM) had a high A/X-ratio (0·82). Their elution profiles showed two polydisperse peaks with apparent MW of respectively 100–120 kDa and 5–10 kDa. Graded ethanol precipitation resulted in a lowly substituted fraction (A/X=0·40; 0–40% ethanol) of high MW and a fraction of highly substituted AX (A/X>0·95; 40–70% ethanol) containing both low molecular weight (LMW-) as well as high molecular weight (HMW-) AX. At an ethanol concentration of 70% or more, only LMW-AX precipitated.

Use of Two Endoxylanases with Different Substrate Selectivity for Understanding Arabinoxylan Functionality in Wheat Flour Breadmaking

ABSTRACTA Bacillus subtilis endoxylanase (XBS) with a strong selectivity for hydrolysis of water‐unextractable arabinoxylan (WU‐AX) and an Aspergillus aculeatus endoxylanase (XAA) with a strong selectivity for hydrolysis of water‐extractable arabinoxylan (WE‐AX) were used in straight‐dough breadmaking with two European wheat flours. Dough, fermented dough, and bread characteristics with different levels of enzyme addition were evaluated with a strong emphasis on the arabinoxylan (AX) population. The WU‐AX solubilized by XBS during breadmaking were mainly released during mixing and had higher molecular weight, in contrast to their counterparts solubilized by XAA, which were mainly released during fermentation and had lower molecular weight. This coincided with increased loaf volume with XBS and a negative to positive loaf volume response with XAA. Bread firmness and dough extract viscosity also were affected by endoxylanase addition. Results confirmed that WU‐AX are detrimental for breadmaking, while WE‐AX and solubilized AX with medium to high molecular weight have a positive impact on loaf volume.

Relative Activity of Endoxylanases Towards Water-extractable and Water-unextractable Arabinoxylan

Differences in endo-β-1,4-xylanase functionality result from the different ability of the enzymes to convert water-unextractable arabinoxylan (WU-AX) into solubilised AX (S-AX) and to degrade water-extractable arabinoxylan (WE-AX) and S-AX to lower molecular weight. These respective endoxylanase activities and their relative proportions were determined for two endoxylanases from Bacillus subtilis and Aspergillus aculeatus, according to a generally applicable procedure. Isolated wheat WE-AX and WU-AX containing wheat squeegee starch were prepared and incubated with different levels of endoxylanases. The supernatants were analysed for reducing xylose (RX) formed and WU-AX solubilised. Molecular weights of the products formed were assessed. Relative activities differed widely for the two endoxylanases. Based on the results and additional enzyme-substrate binding experiments, mechanisms possibly accounting for these differences are discussed.

Determination of reducing end sugar residues in oligo- and polysaccharides by gas–liquid chromatography

Reducing end sugar residues in maltodextrins and arabinoxylans are determined as alditol acetates by gas–liquid chromatography following reduction, acid hydrolysis and acetylation of the samples. After this conversion to alditol acetates, the reducing end sugars are thus separated from their acetylated aldose counterparts. The method allows to identify individual reducing end sugars quantitatively and is a good alternative for colorimetric reducing sugar assays and 1H-NMR analysis. To demonstrate the advantages of the method, an application in a study of enzymic solubilisation and degradation of water unextractable arabinoxylan from a flour squeegee fraction is described.

Milling of Whole Grain Rye to Obtain Fractions with Different Dietary Fibre Characteristics

Rye was separated by dry-milling into three fractions enriched in different rye grain tissue (pericarp/testa, aleurone and endosperm). The fractions varied considerably in gross chemical composition; the starch content ranged from 3·0 g/100 g dm in the pericarp/testa to 78·2 g/100 g dm in the endosperm, and the content of dietary fibre (DF) from 6·5 g/100 g dm in the endosperm to 73·3 g/100 g dm in the pericarp/testa. Protein, fat, ash and phosphorous were concentrated in the aleurone fraction. DF characteristics also differed largely between the rye milling fractions. Arabinoxylans (AX) comprised the majority of DF components in all samples, but on DF basis the milling fractions had different concentrations of lignin, cellulose and β-glucans. Moreover, a wide range in arabinose to xylose ratio of the water-unextractable AX (0·35–1·02) and in the proportion of water-extractable AX (14–70% of total AX) implied that variation in AX structure was obtained. Rye breads intended for a nutritional study were made from the milling fractions. It is concluded that the breads can be used to study the influence of DF characteristics on the colonic fermentation processes in monogastrics.

Distribution of Carbohydrates in Gluten Fractions Isolated from European Wheats (Triticum aestivum L.) in a Batter System

Glucose represented 89% of the total carbohydrate of gluten isolated in a batter system from wheat cultivars exhibiting varying breadmaking qualities. Galactose, arabinose (Ara), and xylose (Xyl) were the remaining carbohydrates (ca. 10% of the total carbohydrate content), while mannose was present in minor quantities (<1%). After Pronase treatment ca. 43% of the Ara and ca. 52% of the Xyl originating from arabinoxylan (AX) could be solubilized, indicating the presence of water extractable as well as water unextractable AX. Arabinogalactan-peptide (AGP) and small amounts of β-glucan were also present. Water extractable AX with a low Ara-to-Xyl ratio (A/X) and thus a low degree of substitution preferentially are incorporated in the gluten network probably as a result of alignment between glutenin polymers and AX in the stirring direction. In gluten from cultivars exhibiting high gluten protein recoveries (see accompanying paper; Roels et al. J. Agric. Food Chem. 1998, 46, 1344−1349), the levels of total gluten associated nonstarch polysaccharides (TOTGANSP), expressed as a percentage of total flour NSP, were also high. It was postulated that the gluten proteins of such cultivars agglomerate despite the presence of NSP since the gluten fractions with the best agglomeration properties contained the lowest amount of NSP. In the aforementioned gluten fractions the AGP/AX ratio was also unusually high, indicating that the undocumented role of AGP in wheat gluten agglomeration may well be underestimated. Keywords: Gluten agglomeration; gluten composition; arabinoxylan; arabinogalactan; Triticum aestivum

Effect of an Enzyme Preparation Containing Pentosanases on the Bread-making Quality of Flours in Relation to Changes in Pentosan Properties

Abstract The effects of a crude pentosanase-containing enzyme preparation were studied on 12 samples of flour varying in quality for French bread making. At the optimal level of addition, the enzyme improved the dough properties, leading to a greater uniformity in quality characteristics and a higher level of quality for all samples. When an excessive amount of enzyme was added, the dough characteristics deteriorated. Changes in the properties of the flour pentosans were studied in the break-making process (kneading, shaping, end of fermentation, baking) using one of the flours, in the absence and presence of pentosanase-containing enzyme preparation at an optimal and excessive levels of addition. Part of the water-unextractable arabinoxylan became extractable during processing of the control sample, but solubilisation occurred to a greater degree with added enzyme. The specific viscosities of water extracts of doughs increased because of the arabinoxylan released from the WUP. Although more arabinoxylan was solubilised at the excessive level of addition, the apparent intrinsic viscosity of dough water-extractable arabinoxylan was greater at the optimum level of addition. These results were essentially confirmed with all the flour samples tested. The effectiveness of the enzyme preparation therefore appeared to be related to the amount and size of the extractable arabinoxylans.

Substitution patterns of water-unextractable arabinoxylans from barley and malt

Intact arabinoxylan polymer fractions from barley and malt differing in their ratio of arabinose to xylose (ara:xyl ratio) were degraded with a purified endoxylanase (E.C. 3.2.1.8) from Aspergillus awamori. Enzymic degradability decreased with increasing ara:xyl ratio. No differences in degradability between corresponding fractions from barley and malt could be observed. The fragments liberated by enzymic action were characterized using HPAEC. Fragments consisting of less than seven pentose residues contained relatively low amounts of O-2- and O-2,3-substituted xylose, compared to the original substrate. Xylose residues substituted at O-2 or both O-2 and O-3 appeared to be concentrated in the larger fragments. From these results and from our knowledge of the mode of action of the xylan degrading enzyme it was concluded that substituted xylose residues were not randomly distributed in the polysaccharide, but were arranged according to a pattern in which isolated unsubstituted residues are separated by one or two substituted residues. This pattern was interrupted by sequences of contiguous unsubstituted xylose residues. From the available data it was concluded that those sequences could reach a length of at least four residues, but the presence of longer unsubstituted sequences could not be excluded. Simulations of arabinoxylan structure and its enzymic degradation supported this model.

Water-unextractable Cell Wall Material from Wheat Flour. 3. A Structural Model for Arabinoxylans

Abstract Water-unextractable wheat flour arabinoxylans were degraded with purified endo-(1→4)-β- d -xylanases and a (1→4)-β- d -arabinoxylan arabinofuranohydrolase from Aspergillus awamori in order to elucidate their structural features. The arabinoxylan fragments obtained were characterized and quantified using Bio-Gel P-2 chromatography in combination with high-performance anion-exchange chromatography. A model is proposed, based on the results obtained, in which arabinoxylans are envisaged as containing highly-branched regions, mostly consisting of tetrameric repeating units of unsubstituted and a double arabinofuranosylated xylose residue, interlinked with less-branched regions, which include subregions containing up to seven contiguous unsubstituted xylose residues. The highly-branched regions are enriched in both O -2,3 disubstituted, as well as O -2 monosubstituted, xylose residues. The latter are absent in the less-branched regions. Variation in arabinose/xylose ratio between different arabinoxylan populations is due to variation in the relative proportion, as well as the composition, of the less-branched regions. In general, water-unextractable arabinoxylans are degraded by the purified xylanases to a lesser extent than water-extractable arabinoxylans with similar arabinose/xylose ratios and glycosidic linkage compositions.